Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to a method and the product obtained thereby of loading therapeutic agents into preformed liposomes, in particular, loading of protonatable compounds by an ammonium ion gradient having glucuronate as the balancing anion.
• PPE palmer-plantar erythrodysestheris
• the onset of PPE may be prevented by prolongation of dosing intervals, however, dose and/or schedule modifications may reduce efficacy against certain tumors, e.g., breast carcinoma (Lyass et al, supra, (2000); Ranson, M.R. et al., J. Clin. Oncol., 15:3185-3191 (1997)).
• the salt precipitates or gels due to its low solubility in the aqueous internal liposomal compartment. This gel formation stabilizes the entrapped doxorubicin in the lipid vesicle and decreases its rate of efflux.
• a method for entrapping therapeutic compounds in preformed liposomes which retains the advantages of the ammonium sulfate gradient, e.g., efficiency and stability, yet enables the entrapped compound to be release at a higher rate would be desirable.
• the invention provides a liposomal composition liposomes comprised of vesicle forming lipids and having an entrapped ionizable therapeutic agent in association with a glucuronate anion.
• the therapeutic agent so loaded has a higher release rate than that loaded by an ammonium gradient having sulfate as the balancing, or counter, anion.
• the vesicle-forming lipids forming the liposomes are phospholipids.
• the liposomes further comprise between about 1 -20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer, such as polyethylene glycol.
• the vesicle-forming lipid is hydrogenated soy phosphatidylcholine (HSPC) and said vesicle-forming lipid derivatized with a hydrophilic polymer is distearoyl phosphatidylethanolamine (DSPE) derivatized with polyethylene glycol.
• HSPC hydrogenated soy phosphatidylcholine
• DSPE distearoyl phosphatidylethanolamine
• the liposomes further comprise cholesterol.
• An exemplary composition is HSPC, cholesterol, and
• the therapeutic agent is an anthracycline antibiotic.
• exemplary anthracycline antibiotic include doxorubicin, daunorubicin, and epirubicin.
• composition described above is used, in another aspect, for treating a patient.
• the composition is used, in another aspect, for treating a neoplasm in a patient.
• the invention includes an improved method of preparing liposomes that have an entrapped ionizable therapeutic agent, where the therapeutic agent is loaded into pre-formed liposomes against an ammonium ion gradient with sulfate as a counterion.
• the improvement comprises loading the ionizable therapeutic agent into liposomes by an ammonium ion gradient having glucuronate as a counterion.
• loading includes preparing a suspension of liposomes, each liposome having at least one internal aqueous compartment that contains ammonium glucuronate at a first concentration, in one embodiment.
• the irrfproved method includes preparing liposomes suspended in an external bulk medium having a second concentration of ammonium glucuronate, wherein the first concentration is higher than the second concentration thereby establishing an ammonium ion concentration gradient across lipid bilayers of the liposomes.
• the improved method includes adding an amount of the therapeutic agent to the suspension of liposomes.
• the invention includes a method of preparing liposomes, comprising forming liposomes having an internal compartment and a bilayer lipid membrane.
• the liposomes have a concentration gradient of ammonium glucuronate across their bilayer lipid membranes.
• The, the liposomes are contacted with an ionizable therapeutic agent to achieve transport of the agent into the internal compartment.
• the method includes (i) preparing a suspension of liposomes, each liposome in the suspension having at least one internal aqueous compartment that contains ammonium glucuronate at a first concentration, the liposomes suspended in an external bulk medium comprising ammonium glucuronate at the first concentration; (ii) reducing the first concentration of ammonium glucuronate in the external bulk medium to a lower, second concentration of ammonium glucuronate, thereby establishing an ammonium ion . concentration gradient across lipid bilayers of the liposomes.
• the step of reducing is achieved by dilution, dialysis, diafiltration, or ion exchange.
• the invention includes a method for loading a protonatable compound into pre-formed liposomes, comprising preparing a suspension of liposomes having a greater concentration of ammonium glucuronate inside the liposomes than outside the liposomes thereby establishing an ammonium ion concentration gradient from the inside to outside of the liposomes.
• the gradient is capable of active transport of said protonatable compound towards the inside of the liposomes.
• the method also includes adding an amount of protonatable compound to the suspension, and allowing the protonatable compound to transport into the liposomes to achieve a content of said protonatable compound inside the liposomes to be greater than that outside of the liposomes.
• the method includes forming the liposomes in the presence of an ammonium glucuronate solution having a first concentration; and entrapping said ammonium glucuronate solution of said first concentration inside said liposomes; and reducing said first concentration of said ammonium glucuronate solution outside of the liposomes to a second concentration which is less than that of said first concentration.
• the method of the invention has a high loading efficiency. In one embodiment greater than 50% of the amount of protonatable compound added to the suspension is transported to the inside of the liposomes. In another embodiment approximately 90% of the amount of protonatable compound added to the suspension is transported to the inside of the liposomes. In specific embodiments, the loading efficiency for doxorubicin is greater than 90% and the doxorubicin to phospholipid ratio is in the range of about 100- 150 ⁇ g/ ⁇ mol.
• Figs. 1A-1E are growth inhibition curves plotting the growth rate, as a percent of untreated control cells, of mouse cell lines M109ST (Fig. 1A), M109R (Fig. 1B) and of human cell lines C-26 (Fig. 1C), KB (Fig. 1D), and KB-V (Fig.
• Fig. 3 is a bar graph showing doxorubicin concentration ( ⁇ g/mL) in mouse plasma at various times after the injection of liposomes containing doxorubicin, where the doxorubicin was remotely loaded into the liposomes against an ammonium sulfate gradient (hatched bars) or against an ammonium glucuronate gradient (dotted bars);
• Fig. 4 is a plot of mean footpad thickness, in mm, in mice inoculated with M109-S cells as a function of days after treatment with saline (closed squares), free doxorubicin (circles), or doxorubicin entrapped in liposomes, where the doxorubicin was remotely loaded into the liposomes against an ammonium sulfate gradient (triangles, "lipo-dox-AS") or against an ammonium glucuronate gradient (open squares, "lipo-dox-AG”);
• Fig. 5 is a plot of mean footpad thickness, in mm, in mice inoculated with M109R cells (doxorubicin-resistant tumor cells) as a function of days after treatment with saline (closed squares), free doxorubicin (circles), or doxorubicin entrapped in liposomes, where the doxorubicin was remotely loaded into the liposomes against an ammonium sulfate gradient (triangles, "lipo-dox-AS") or against an ammonium glucuronate gradient (open squares, "lipo-dox-AG”); and [0028] Fig.
• FIG. 6 is a plot of number of surviving mice as a function of days after inoculation with C-26 tumor cells and treatment with free doxorubicin (circles) or with doxorubicin entrapped in liposomes, where the doxorubicin was remotely loaded into the liposomes against an ammonium sulfate gradient (triangles, "lipo- dox-AS") or against an ammonium glucuronate gradient (squares, "iipo-dox-AG”).
• the invention provides a liposomal compositon where an ionizable therapeutic agent is entrapped in the internal liposomal compartment(s) in the form of an ionic salt with monovalent glucuronate anions.
• the entrapped therapeutic agent has a faster release rate from the liposomes compared to the release rate of the agent entrapped in the liposomes in the form of an ionic salt with divalent sulfate anions.
• the invention also provides a remote loading procedure for loading therapeutic agents into pre-formed liposomes against an ammonium glucuronate gradient. The faster rate of release of the therapeutic agent from the liposomes affords flexibility to adjust dosing schedules without compromising the biological efficacy of the therapeutic agents.
• the method of the invention therefore provides a beneficial alternative to loading by ammonium sulfate.
• the ammonium glucuronate remote loading method does not require the liposomes to be prepared in acidic pH, nor to alkalinize the extraliposomal aqueous medium.
• the approach also permits the loading of therapeutic agents in a broad spectrum of liposomes of various types, sizes, and compositions, including sterically- stabilized liposomes, immunoliposomes, and sterically-stabilized immunoliposomes.
• "Entrapped" as used herein refers to an agent entrapped within the aqueous spaces of the liposomes or within the lipid bilayers.
• the higher release rate is a result of using glucuronate as the balancing anion.
• glucuronate ion being monovalent and containing several hydroxyl functional groups on its six-membered ring, is less effective compared to a sulfate ion at inducing aggregation and precipitation of the therapeutic agent after being transported inside the liposomes.
• the inventors have observed that the solubility of doxorubicin is approximately 100-fold greater in a 250 M ammonium glucuronate (AG) solution than in a 250 mM ammonium sulfate (AS) solution.
• doxorubicin precipitates at less than 2 mM concentration in the presence of sulfate ions, while a much higher concentration of doxorubicin is required for precipitation to occur in the presence of glucuronate ions. Accordingly, when glucuronate is the balancing anion, more of the therapeutic agent is in a soluble form and therefore it is more available for release from the liposomes. Further, the permeability of glucuronate through the liposomal membranes is very low, possibly due to its low pKa, its bulkiness and/or polarity, making it very efficient for maintaining the ammonium ion gradient for loading of the therapeutic agents.
• the method of the invention can be used to remotely load essentially any therapeutic agent which is protonatable (can exist in a positively charged state) when dissolved in an appropriate aqueous medium.
• the agent should be relatively lipophilic so that it will partition into the lipid vesicle membranes.
• the therapeutic compound for loading is a weak amphipathic compound, that is a compound having either weak basic or acidic moieties.
• therapeutic agents which can be loaded into liposomes by the method of the invention include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, streptozocin, vinblastine, vincristine, mechlorethamine hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide, carmustine, lomustine, semustine, fluoruracil, hydroxyurea, thioguanine, cytarabine, floxuridine, decarbazine, cisplatin, procarbazine, ciprofloxacin, epirubicin, carcinomycin, N-acetyladriamycin, rubidazone, 5-imidodaunomycin, N- acetyldaunomycine, all anthracyline drugs, daunoryline, propranolol, pentamindine, dibucaine, tetracaine,
• the method can be used to load multiple therapeutic agents, either simultaneously or sequentially.
• the liposomes into which the protonatable therapeutic agents are loaded can themselves be pre-loaded with other pharmaceutical agents or drugs using conventional encapsulation techniques (e.g., by incorporating the drug in the buffer from which the liposomes are prepared).
• the method of the invention therefore provides great flexibility in preparing liposome encapsulated "drug cocktails" for use in therapies.
• one or more of the protonatable drugs listed above can be pre-loaded and then the same or a different drug can be added to the liposomes using the ammonium glucuronate gradient of the present invention.
• the method is particularly suitable for loading weakly amphipathic drugs such as doxorubicin.
• Doxorubicin loaded in liposomes having an external surface coating of hydrophilic polymer chains by an ammonium glucuronate gradient (referred to herein as "lipo-dox-AG”) exhibits a faster release rate than doxorubicin loaded in liposomes having an external surface coating of hydrophilic polymer chains by an ammonium sulfate gradient (referred to herein as "lipo-dox- AS"; commercially known as Doxil ® ), and has similar biological efficacy.
• Liposomes suitable for use in the compositions of the present invention include those composed primarily of vesicle-forming iSpids. Vesicle-forming lipids, exemplified by the phospholipids, form spontaneously into biiayer vesicles in water at physiological pH and temperatures.
• the liposomes can also include other lipids, incorporated into the lipid bilayers, with the hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and the head group moiety oriented toward the exterior, polar surface of the bilayer membrane.
• the vesicle-forming lipids are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar.
• diacyl synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids such as phospholipids, diglycerides, dialiphatic glycolipids, single lipids such as sphingomyelin and glycosphingolipid, cholesterol and derivatives thereof, alone or in combinations and/or with or without liposome membrane rigidifying agents.
• phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin, plasmalogens, and phosphatidylcholine lipid derivatives where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
• the above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods.
• Cationic lipids are also suitable for use in the liposomes of the invention, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component.
• Such cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge.
• the head group of the lipid carries the positive charge.
• Exemplary cationic lipids include 1 ,2-dioleyloxy-3- (trimethylarnino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-NN ⁇ dimethyl-N-hydroxyethylanimonium bromide (DMRIE); N-[l-(2,3,- dioleyloxy)propyl]-NN-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N- [1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 30[N- (N'.N'-dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethyldioctadecylammonium (DDAB).
• DOTAP 1,2-dioleyloxy-3- (trimethylarnino) propane
• DMRIE dimethyl-N-hydroxyethylanimonium bro
• the cationic vesicle-forming lipid may also be a neutral lipid, such as dioleoylphosphafidyl ethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic lipid, such as polylysine or other polyarnine lipids.
• DOPE dioleoylphosphafidyl ethanolamine
• an amphipathic lipid such as a phospholipid
• a cationic lipid such as polylysine or other polyarnine lipids.
• the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid.
• the vesicle-forming lipid can be selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in serum and to control the rate of release of the entrapped agent in the liposome.
• Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., above room temperature, more preferably above body temperature and up to 80°C.
• Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer.
• Other lipid components, such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures.
• Lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature, more preferably, at or below body temperature.
• a relatively fluid lipid typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature, more preferably, at or below body temperature.
• the liposomes may optionally include a vesicle-forming lipid derivatized with a hydrophilic polymer, as has been described, for example in U.S. Patent No. 5,013,556 and in WO 98/07409, which are hereby incorporated by reference.
• Incorporation of a hydrophilic polymer-lipid conjugate into the liposomal bilayer polymer provides a surface coating of hydrophilic polymer chains on both the inner and outer surfaces of the liposome lipid bilayer membranes.
• the outermost surface coating of hydrophilic polymer chains is effective to extend the blood circulation lifetime in vivo relative to liposomes lacking the polymer chain coating.
• the inner coating of hydrophilic polymer chains extends into the aqueous compartments in the liposomes, i.e., between the lipid bilayers and into the central core compartment, and is in contact with any entrapped agents.
• Vesicle-forming lipids suitable for derivatization with a hydrophilic polymer include any of those lipids listed above, and, in particular phospholipids, such as distearoyl phosphatidylethanolamine (DSPE).
• Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylainide, polydirnethylacrylamide, polyhydroxypropyhnethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
• the polymers may be employed as homopolymers or as block or random copolymers.
• a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between about 500 and about 10,000 Daltons, more preferably between about 500 and about 5,000 Daltons, most preferably between about 1 ,000 to about 2,000 Daltons.
• PEG polyethyleneglycol
• Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., 120-20,000 Daltons.
• Preparation of vesicle-forming lipids derivatized with hydrophilic polymers has been described, for example in U.S. Patent No. 5,395,619.
• liposomes including such derivatized lipids have also been described, where typically, between 1-20 mole percent of such a derivatized lipid is included in the liposome formulation.
• the hydrophilic polymer may be stably coupled to the lipid, or coupled through an unstable linkage which allows the coated liposomes to shed the coating of polymer chains as they circulate in the bloodstream or in response to a stimulus, as has been described, for example, in U.S. Patent No. 6,043,094, which is incorporated by reference herein.
• Liposomal suspensions comprised of liposomes having an ion gradient across the liposome bilayer (also referred to as a 'transmembrane gradient') for use in remote loading can be prepared by a variety of techniques, such as those detailed in Szoka, F., Jr., et al., Ann Rev Biophys Bioeng 9:467, (1980).
• Multilarnellar vesicles (MLVs) can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids of the type described above is dissolved in a suitable organic solvent and the solvent is later evaporated off leaving behind a thin film.
• the film is then covered by an aqueous medium, containing the solute species, e.g., ammonium glucuronate, which forms the aqueous phase in the liposome interior spaces and also the extraliposomal suspending solution.
• the lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
• the lipids used in forming the liposomes of the present invention are preferably present in a molar ratio of about 70-100 mole percent vesicle-forming lipids, optionally 1-20 mole percent of a lipid derivatized with a hydrophilic polymer chain.
• One exemplary formulation includes 80-90 mole percent phosphatidylethanolamine, 1-20 mole percent of PEG-DSPE. Cholesterol may be included in the formulation at between about 1-50 mole percent.
• the lipid components are hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Choi) and methoxy-capped polyethylene glycol derivatized distearyl phosphatidylethanolamine (mPEG(2000)-DSPE) in a molar ratio of 92.5:70:7.5.
• HSPC hydrogenated soy phosphatidylcholine
• Choi cholesterol
• mPEG(2000)-DSPE methoxy-capped polyethylene glycol derivatized distearyl phosphatidylethanolamine
• the hydration medium contains ammonium glucuronate.
• concentration of ammonium glucuronate would depend on the amount of therapeutic agent to be loaded. Typically, the concentration is between 100 to 300 mM of ammonium glucuronate. In one preferred embodiment, the hydration medium contains 250 mM ammonium glucuronate.
• the vesicles formed by the thin film method may be sized to achieve a size distribution within a selected range, according to known methods.
• the liposomes are uniformly sized to a size range between 0.04 to 0.25 ⁇ m.
• Small unilamellar vesicles (SUVs) typically in the 0.04 to 0.08 ⁇ m range, can be prepared by post-formation sonication or homogenization.
• Homogeneously sized liposomes having sizes in a selected range between about 0.08 to 0.4 ⁇ m can be produced, e.g., by extrusion through polycarbonate membranes or other defined pore size membranes having selected uniform pore sizes ranging from 0.03 to 0.5 ⁇ m, typically, 0.05, 0.08, 0.1 , or 0.2 ⁇ m.
• the pore size of the membrane corresponds roughly to the largest size of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane.
• the sizing is preferably carried out in the original lipid-hydrating buffer, so that the liposome interior spaces retain this medium throughout the initial liposome processing steps.
• a therapeutic agent is loaded into the liposomes after sizing.
• a "remote” or “active” loading process results from exchange of the therapeutic agent in the external or bulk medium in which the liposomes are suspended with an ammonium ion in internal liposomal compartment.
• the efficiency of loading depends, at least in part, on an ammonium ion gradient, where the concentration of the ammonium ion inside the liposomes is higher than the concentratration of ammonium ion in the external, bulk suspension medium. The magnitude of this gradient determines to a large extent the level of encapsulation; the larger the gradient, generally the higher the encapsulation.
• An ammounium glucuronate gradient across the liposomal lipid bilayer, where the ammonium ion concentration is higher on the inside of the liposomes than in the external suspension medium may be formed in a variety of ways, e.g., by (i) controlled dilution of the external medium, (ii) dialysis against the desired final medium, (iii) molecular-sieve chromatography, e.g., using Sephadex G-50, against the desired medium, or (iv) high-speed centrifugation and resuspension of pelleted liposomes in the desired final medium.
• the final external medium selected will depend on the mechanism of gradient formation and the external ion concentration desired.
• the gradient is measured as the ratio of ammonium glucuronate inside to that outside of the liposomes. Generally, the gradient is in the range of 1000-10 inside/outside. Preferably, the gradient is in the range of 500-50.
• the concentration of ammonium glucuronate in an external medium that also contains electrolytes may be measured as ammonia concentration at pH 13- 14 (Bolotin, E.M., et al., Journal of Liposome Research 4(l):455-479 (1994)) by an ion analyzer, e.g., a Coming 250 pH/ion analyzer (Corning Science Products, Corning, NY) equipped with a Corning 476130 ammonia electrode and an automatic temperature compensation (ATC) stainless steel probe, if the final external medium lacks electrolytes the ammonium glucuronate gradient may be confirmed by conductivity measurements using a conductivity meter, e.g., a type CDM3 conductivity meter equipped with a CDC 304 immersion electrode with manual temperature compensator type CDA 100 (Radiometer, Copenhagen, Denmark).
• a conductivity meter e.g., a type CDM3 conductivity meter equipped with a CDC 304 immersion electrode with manual temperature compensator type CDA 100
• the ammonium ion gradient is created by controlled dilution.
• This method gives a diluted liposome preparation.
• the liposomal suspension has a selected first concentration of ammonium glucuronate inside the liposome and in the external bulk medium.
• the external bulk medium is diluted with a second medium containing no ammonium glucuronate.
• Exemplary second medium include aqueous solutions containing electrolytes (sodium chloride or potassium chloride) or aqueous solutions containing non electrolytes (glucose or sucrose).
• the internal and external media are preferably selected to contain about the same osmolarity, e.g., by suitable adjustment of the concentration of buffer, salt, or low molecular weight solute, such as sucrose.
• a preferred second medium is 15 mM HEPES buffer containing 5% dextrose at approximately pH 7.
• a proton gradient across the lipid bilayer is produced by dialysis in which the external bulk medium is exchanged for one lacking ammonium ions, e.g., the same buffer but one in which ammonium glucuronate is replaced by a salt such as NaCI or KCI, or by a sugar that gives the same osmolarity inside and outside of the liposomes.
• the gradient can be created by four consecutive dialysis exchanges against 25 volumes of the dialysis buffer.
• the gradient may be prepared by a three-step tangential flow dialysis, e.g., using a Minitan ultrafiltration system (Millipore Corp., Bedford, MA) equipped with "300 K" polysulfone membranes.
• the dialysis buffer contain electrolytes (e.g., sodium chloride or potassium chloride) or non electrolytes (glucose or sucrose).
• the dialysis buffer is 15 mM HEPES containing 5% dextrose at approximately pH 7.
• Ammonia gas is permeable in the lipid bilayer, with a permeability coefficient of around 1.3 x 10 "1 cm/second, and is able to permeate the liposomal bilayer.
• the efflux of ammonia shifts the equilibrium within the liposome towad production of protons which results in a [H + ] gradient, with the intraliposomal concentration higher than that in the extraliposomal medium.
• Unprotonated drug crosses the liposomal bilayer, becomes protonated inside the liposome, and is stabilized by the anions present in the internal aqueous compartment of the liposome. Formation of a drug-glucuronate salt elevates the intraliposomal pH and induces formation of NH 3 inside the liposmes.
• a therapeutic agent e.g., doxorubicin
• doxorubicin may be loaded into the liposomes by adding a solution of the agent to a suspension of liposomes having an ammonium ion gradient across the liposomal membranes. The suspension is treated under conditions effective to allow passage of the compound from the external medium into the liposomes.
• Incubation conditions suitable for drug loading are those which (i) allow diffusion of the compound, which is in an uncharged form, into the liposomes, and (ii) preferably lead to high drug loading concentration, e.g., 5-500 mM drug encapsulated, more preferably between 20-300 mM, most preferably between 50-200 mM.
• high drug loading concentration e.g., 5-500 mM drug encapsulated, more preferably between 20-300 mM, most preferably between 50-200 mM.
• the loading is preferably carried out at a temperature above the phase transition temperature of the liposome lipids.
• the loading temperature may be as high as 60"C or more.
• the loading period is typically between 15-120 minutes, depending on permeability of the drug to the liposome bilayer membrane, temperature, and the relative concentrations of liposome lipid and drug. In one preferred embodiment, the loading is performed at 60°C and for 60 minutes.
• encapsulation of doxorubicin can be greater than 90%. Knowing the calculated internal liposome volume, and the maximum concentration of loaded drug, one can then select an amount of drug in the external medium which leads to substantially complete loading into the liposomes.
• the liposome suspension may be treated, following drug loading, to remove non-encapsulated drug.
• Free drug can be removed, for example, by ion exchange chromatography, molecular sieve chromatography, dialysis, or centrifugation.
• the non-entrapped drug is removed using Dowex 50WX-4 (Dow Chemical, Ml).
• free doxorubicin but not liposomal doxorubicin
• a cation exchange resin Storm, G. etal, Biochim Biophys Acta, 818:343 (1985)
• Table 1 shows the doxorubicin concentration needed to inhibit 50% of cell grow (IC50 values) for free doxorubicin (F-DOX), lipo-dox-AG, and lipo-dox- AS.
• Doxorubicin in free from is more cytotoxic than either of the two liposomal doxorubicin formulations.
• Doxorubicin loaded into liposomes against an ammonium glucuronate gradient is more cytotoxic than when loaded into liposomes against an ammonium sulfate gradient, suggesting that the drug is more bioavailable from a glucuronate salt than from a sulfate salt.
• Figs. 1A-1E show the inhibition curves for the mouse cell lines, M109-S (Fig. 1A), M109-R (Fig. 1B) and the human cell lines C-26 (Fig. 1C), KB (Fig. 1D), and KB-V (Fig. 1 E).
• the doxorubicin concentration, in nM, of the different formulations are represented as free doxorubicin (circles), lipo-dox-AG (squares), and lipo-dox-AS (triangles). All the drug formulations at doxorubicin concentrations between 10 2 to 10 6 were cytotoxic to each of the tumor cell lines tested. In all cases, with variations in the growth rate inhibition, lipo-dox-AG was more cytotoxic than lipo-dox-AS, showing that drug from the liposomal-ammonium glucuronate platform was more readily bioavailable than drug from the liposomal-ammonium sulfate platform.
• doxorubicin entrapped in liposomes by loading against an ammonium glucoronate gradient were evaluated in 3-month-old BALB/c female mice.
• liposomes with entrapped doxorubicin loaded against ammonium sulfate or ammonium glucuronate were injected intravenously into the mice. Blood samples were taken at selected intervals and analyzed for doxorubicin concentration.
• Fig. 3 shows the plasma doxorubicin concentration for mice treated with lipo-dox-AG (cross hatched bars) or with lipo-dox-AS (dotted bars).
• the half-life of doxorubicin when administered from a lipo-dox-AG platform is approximately 16 hours, while that of doxorubicin when administered from a lipo-dox-AS platform is approximately 24 hours. It is also apparent that lipo-dox-AG is cleared faster than lipo-dox-AS.
• the lipo-dox- AG blooc concentrations were 25% lower at 4 hours post intravenous administration, 33% lower at 24 hours, and almost 50% lower at 48 hours post intravenous administration. Since the composition and size of the liposomes were identical, the rate of uptake by the reticuloendothelial system (RES.) should be similar. Accordingly, the faster clearance is probably the result of a faster release rate in vivo of doxorubicin from the lipo-dox-AG formulation, consistent with the the In vitro experiments.
• RES. reticuloendothelial system
• mice were inoculated with M109S tumor cells (10 6 cells) and treated with a single dose of doxorubicin at 10 mg/kg of either free doxorubicin, lipo-dox-AS, or lipo-dox-AG post tumor inoculation.
• mice were inoculated with M109R cells (10 6 cells). Ten days after inoculation, the mice were treated with either free doxorubicin, lipo-dox-AS, or lipo-dox-AG at a dose of 8 mg/kg. The same dose was administered again one week and three weeks later.
• mice were inoculated with C-26 cells (10 6 cells) to induce a tumor and treated, five days after tumor inoculation, with either free doxorubicin, lipo-dox-AS, or lipo-dox-AG at a doxorubicin dose of 10 mg/kg.
• Fig. 6 shows the number of surviving mice as a function of time post tumor inoculation. Untreated (control) mice died quickly with a median survival of 13 days (not shown). Mice treated with free doxorubicin (circles) showed a neglible increase in mean survival time (4 days more than control, i.e., 17 days). Both liposomal preparations (squares, triangles) were more effective in extending survival time in tumor-bearing mice than was free doxorubicin.
• Liposomes having a drug entrapped in the form of a glucuronate salt provide a higher release rate of drug than does a similar liposome where the drug is entrapped in the form of a sulfate salt, without significant effect on drug efficacy.
• Clinical data with liposome-entrapped doxorubicin (Doxil ® ) indicate that the incidence and severity of PPE decrease with a shortening of the circulation half-life of Doxil ® , the faster release, and shorter circulation of doxorubicin in the form of lipo-dox-AG provides a good alternative for doxorubicin delivery. It will be appreciated that the findings specific to doxorubicin extend to other drugs capable of remote loading against an ammonium ion gradient, such as those recited herein.
• Liposome Preparation and Loading A. Liposome Preparation [0072] Liposomes containing ammonium glucuronate in the aqueous compartments were prepared as follows. The lipid component, hydrogenated soy phosphatidylcholine (HSPC), cholesterol and methoxy-capped polyethylene glycol derivatized distearyl phosphatidylethanolamine (mPEG(200)-DSPE) in a molar ratio of 92.5:70:7.5, were dissolved in chloroform. The solvent was evaporated using a rotary evaporator under reduced pressure leaving behind a dried lipid thin film.
• HSPC hydrogenated soy phosphatidylcholine
• mPEG(200)-DSPE methoxy-capped polyethylene glycol derivatized distearyl phosphatidylethanolamine
• the dried lipid thin film was hydrated with a 250 mM aqueous ammonium glucuronate buffer solution (pH 5.5), forming liposomes containing ammonium glucuronate in the internal aqueous compartments and suspended in an ammonium glucuronate external bulk medium .
• the liposomes were then sized by extrusion through 0.5 ⁇ m pore size membranes.
• a comparative liposome formulation containing 250 mM ammonium sulfate in the interior aqueous compartments was similarly prepared by using 250 ⁇ m ammonium sulfate as the hydration buffer. The batches obtained were similar to the ammonium glucuronate preparations in vesicle size, drug-loading efficiency, and drug-to-phospholipid ratio.
• Free doxorubicin i.e., doxorubicin not entrapped in a liposome
• Free doxorubicin i.e., doxorubicin not entrapped in a liposome
• EXAMPLE 2 In vitro Characterization A. In vitro Cytotoxicity [0077] Free doxorubicin and liposomal formulations of doxorubicin, prepared as described above in Example 1 , were tested against five mouse and human tumor cell lines (M109-S, M109-R, C-26, KB, KB-V).
• the cultures were fixed by the addition of 50 ⁇ L 2.5% glutaraldehyde to each well for 10 minutes.
• the plates were washed three times with de-ionized water, once with 0.1 M borate buffer (pH 8.5) and then stained for 60 minutes with 100 ⁇ L methylene blue (1% in 0.1 M buffer borate, pH 8.5) at room temperature.
• the plates were rinsed in five baths of de-ionized water to remove non-cell bound dye and were then dried.
• the dye was extracted with 200 ⁇ L 0.1 M HCl for 60 min at 37°C and the optical density was determined using a microplate spectrophotometer.
• the growth rate was calculated by dividing the doubling times of drug- treated cells with those of the control cells.
• the drug concentration which caused a 50% inhibition of the control growth rate (IC50) was calculated yb interpolation of the two closest values of the growth inhibition curve.
• Table 1 shows the IC50 values for free doxorubicin, lipo-dox-AS, and lipo-dox-AG for each of the cell lines, and the corresponding growth inhibition curves are shown in Figs. 1A-1 E.
• the cells were rinsed three times with PBS and the drug was extracted from the cells with 1 mL acidified isopropanol (0.075 M HC1 in 90% isopropanol), for 20 hours at 4°C.
• Doxorubicin concentration was determined spectrofluorometrically using an excitation wavelength of 470 nm and an emission wavelength of 590 nm. The fluorescence intensity emitted was translated into doxorubicin-equivalents based on a doxorubicin standard curve, after readings of untreated background cells were subtracted.
• mice Thirty mice were inoculated in the footpad with M109-S cells (10 6 cells). Seven days later, when the footpad thickness increased from a normal value of approximately 1.5 mm to an average of 2.0-2.5 mm, the mice were divided into three groups of 10 each and the mice groups were injected intravenously with either free doxorubicin, lipo-dox-AS, or lipo-dox-AG at a doxorubicin dose of 10 mg/kg. Thereafter, the footpad thickness was measured twice a week with alipers to follow tumor growth and effect of therapy. The results are shown in Fig. 4.
• mice were inoculated in the footpad with the doxorubicin-resistant tumor cell line M109R cells (10 6 cells). Ten days later, when the footpad thickness increased from a normal value of approximately 1.5 mm to an average of 2.0-2.5 mm, the mice were divided into three groups for intravenous treatment with free doxorubicin, lipo-dox-AS, or lipo-dox-AG at a doxorubicin dose of 8 mg/kg. Two additional injections were given at the same dose 1 week and 3 weeks later. The footpad thickness was measured twice a week with calipers and the results are shown in Fig. 5.
• mice were inoculated i.p. with C-26 cells (10 6 cells). Five days later, the mice were separated into three groups of 10 mice each, and each group of mice was injected intravenously with either free doxorubicin, lipo- dox-AS, or lipo-dox-AG at a dose of 10 mg/kg. The survival of these mice was followed and survival curves are shown in Fig. 6.

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